The present invention relates to novel polypeptides derived from bee venom and methods of use thereof, including pharmaceutical compositions useful for modulating immune responses to bee pollen.
Bee venom (BV) is a complex mixture of antigens that can include one or more toxic polypeptides. Many of these polypeptides are hypersentizing agents and can additionally have hemolytic or neurotoxic effects.
Some individuals are hypersensitive to BV polypeptides. IgE antibodies from BV hypersensitive individuals recognize several BV toxic polypeptides. BV polypeptides, often referred to as allergens, recognized by IgE in BV hypersentive individuals can include, e.g., phospholipase A2 (PLA2), acid phosphatase, hyaluronidase, allergen C, and other, high molecular weight (MW) proteins.
BV hypersensitive individuals can be at high risk of an adverse reaction to a bee sting. One recognized method for preventing or minimizing serious adverse reactions resulting from a bee sting is to desensitize the individual to the allergens present in BV. This protection can be induced by a process termed venom immunotherapy (VIT).
The invention is based, in part, on the discovery of a novel bee venom protein, which has been named Api m 6. Polypeptides derived from Api m 6 polypeptides can be used, e.g., in venom immunotherapy to protect susceptible individuals from the adverse effects of a bee sting.
Accordingly, in one aspect, the invention provides a substantially pure polypeptide comprising an amino acid sequence at least 70% identical, and more preferably at least 90% identical, to the amino acid sequence of an Api m 6 polypeptide, e.g., a peptide which includes the amino acid sequence of SEQ ID NO:1. In some embodiments, the polypeptide binds to a human IgE antibody.
In other embodiments, the polypeptide includes a sequence at least 70% identical to one or more of the amino acid sequences of SEQ ID NOs:2-4. Preferably, the polypeptide has an amino acid sequence corresponding to the amino acid sequence of an Apis spp. bee venom protein, e.g., an Apis mellifera bee venom protein. In some embodiments, the polypeptide is glycosylated.
The polypeptide may, in some embodiments, stimulate T-cell proliferation.
The invention also features an antibody that binds to an Api m 6 protein. The antibody can be a polyclonal or a monoclonal antibody.
The invention also features a composition comprising polypeptide fragments of the Api m 6 protein, wherein the polypeptide fragments are between 6-72 amino acids in length. In preferred embodiments, the polypeptide fragments are between 20-100, 30-70, or 40-60 amino acids in length. Preferably, at least one polypeptide in the composition has an amino acid sequence that overlaps by at least 3 amino acids with at least one other polypeptide in the composition, e.g., polypeptide fragments of Api m 6 that overlap by between 5 and 10 amino acids. In the most preferred embodiments, the composition comprises of a set of polypeptide fragments that map the total length of the Api m 6 protein.
The invention also features a pharmaceutical composition that includes an Api m 6 polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes an additional polypeptide, e.g., a second, third, fourth, or more bee venom polypeptide or polypeptides.
The additional bee venom polypeptides can include, e.g., phospholipase A2, hyaluronidase, allergen C, mellitin, adolapin, minimine, acid phosphatase, protease inhibitor, and glycosylated IgE-binding proteins, or analogs or derivatives thereof.
In another aspect, the invention features a method of modulating an immune response. The method includes administering an Api m 6 polypeptide to a subject in need thereof in an amount sufficient to inhibit an immune reaction by the subject against the polypeptide. If desired, one or more additional bee venom polypeptides may also be administered to the subject. The additional bee venom polypeptides can include, e.g., phospholipase A2, hyaluronidase, allergen C, mellitin, adolapin, minimine, acid phosphatase, protease inhibitor, and glycosylated IgE-binding proteins, or analogs or derivatives thereof.
In a further aspect, the invention includes a method of identifying an individual at risk for bee venom hypersensitivity. The method includes administering to the individual an Api m 6 polypeptide and measuring an immune response raised against the polypeptide. A detectable immune response indicates that the individual is at risk for bee venom hypersensitivity. In preferred embodiments, the Api m 6 polypeptide is administered intradermally. Preferably, the Api m 6 polypeptide is administered at a concentration of less than about 1 xcexcg/ml.
Also provided is a method of purifying an Api m 6 polypeptide. The method includes providing a cell expressing the Api m 6 polypeptide. The cell is then contacted with an antibody which binds to a polypeptide that includes an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO:1 under conditions which allow for formation of a polypeptide-antibody complex. The antibody-polypeptide complex is then isolated, and the Api m 6 polypeptide is recovered from the complex.
Also provided by the invention is a kit that includes, in one or more containers, an Api m 6 polypeptide, overlapping polypeptide fragments of an Api m 6 polypeptide, an anti Api m 6 polypeptide antibody (e.g., a monoclonal or polyclonal antibody), or a combination of these polypeptides or antibodies.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.